Poly(vinyl alcohol)-Incorporated Core-Shell Polymer Nanogels Functionalized by Block Copolymer Installation for Cisplatin Delivery.

The functionalization approach for nanomaterials is of great importance for their application in drug delivery systems. Herein, an approach based on block copolymer installation into polymer nanogels was newly developed. Poly(vinyl alcohol)-incorporated polymer nanogels were prepared by a two-step dispersion/precipitation polymerization. Poly(methacrylic acid)-block-poly(3-fluorophenylboronic acid methacrylamide) (PMAA-b-PFPBMA) prepared by two-step reversible addition-fragmentation chain transfer polymerization was installed into the polymer nanogels via boronate ester formation. Furthermore, cisplatin as a cancer therapeutic drug was successfully loaded on the block copolymer-installed polymer nanogels, and cell death was achieved by using the resulting cisplatin-loaded nanogels. We believe that the functionality of the nanogels can be changed by varying the installed block copolymer, leading to the functionalization approach of polymer nanogels based on block copolymer installation, which will be of great utility in many fields.

Effect of Poly(Vinyl Alcohol) Concentration and Chain Length on Polymer Nanogel Formation in Aqueous Dispersion Polymerization.

Nanotechnology has attracted increasing interest in various research fields for fabricating functional nanomaterials. In this study, we investigated the effect of poly(vinyl alcohol) (PVA) addition on the formation and thermoresponsive properties of poly(N-isopropyl acrylamide)-based nanogels in aqueous dispersion polymerizations. During dispersion polymerization, PVA appears to play three roles: (i) it bridges the generated polymer chains during polymerization, (ii) it stabilizes the formed polymer nanogels, and (iii) it regulates the thermoresponsive properties of the polymer nanogels. By regulating the bridging effect of PVA via changing the PVA concentration and chain length, the size of the obtained polymer gel particles was maintained in the nanometer range. Furthermore, we found that the clouding-point temperature increased when using low-molecular weight PVA. We believe that the knowledge gained in this study regarding the effect of PVA concentration and chain length on nanogel formation will aid in the future fabrication of functional polymer nanogels.

Programed Thermoresponsive Polymers with Cleavage-Induced Phase Transition.

A new programed upper critical solution temperature-type thermoresponsive polymer was developed using water-soluble anionic polymer conjugates derived from polyallylamine and phthalic acid with cleavage-induced phase transition property. Intrinsic charge inversion from anion to cation of the polymer side chain is induced through a side chain cleavage reaction in acidic aqueous media. With the progress of side chain cleavage under fixed external conditions, the polymer conjugates express a thermoresponsive property, followed by shifting a phase boundary due to the change in polymer composition. When the phase transition boundary eventually reached the examined temperature, phase transition occurs under fixed external conditions. Such new insight obtained in this study opens up the new concept of time-programed stimuli-responsive polymer possessing a cleavage-induced phase transition.

Antibody-Conjugated Signaling Nanocavities Fabricated by Dynamic Molding for Detecting Cancers Using Small Extracellular Vesicle Markers from Tears.

Small extracellular vesicles (sEVs) are reliable biomarkers for early cancer detection; however, conventional detection methods such as immune-based assays and microRNA analyses are not very sensitive and require sample pretreatments and long analysis time. Here, we developed a molecular imprinting-based dynamic molding approach to fabricate antibody-conjugated signaling nanocavities capable of size recognition. This enabled the establishment of an easy-to-use, rapid, sensitive, pretreatment-free, and noninvasive sEV detection platform for efficient sEV detection-based cancer diagnosis. An apparent dissociation constant was estimated to be 2.4 × 10-16 M, which was ∼1000 times higher than that of commercial immunoassays (analysis time, 5 min/sample). We successfully used tears for the first time to detect cancer-related intact sEVs, clearly differentiating between healthy donors and breast cancer patients, as well as between samples collected before and after total mastectomy. Our nanoprocessing strategy can be easily repurposed for the specific detection of other types of cancer by changing the conjugated antibodies, thereby facilitating the establishment of liquid biopsy for early cancer diagnosis.

Molecularly Imprinted Nanogels Capable of Porcine Serum Albumin Detection in Raw Meat Extract for Halal Food Control.

Accurate, simple, and valuable analytical methods for detection of food contamination are rapidly expanding to evaluate the validity of food product quality because of ethnic considerations and food safety. Herein molecularly imprinted nanogels (MIP-NGs), capable of porcine serum albumin (PSA) recognition, were prepared as artificial molecular recognition elements. The MIP-NGs were immobilized on a quartz crystal microbalance (QCM) sensor for detection of pork contamination in real beef extract samples. The MIP-NGs-based QCM sensor showed high affinity and excellent selectivity toward PSA compared to reference serum albumins from five different animals. The high PSA specificity of MIP-NGs led to the detection of pork contamination with a detection limit of 1% (v/v) in real beef extract samples. We believe the artificial molecular recognition materials prepared by molecular imprinting are a promising candidate for halal food control.

Molecularly Imprinted Nanogels Possessing Dansylamide Interaction Sites for Controlling Protein Corona In Situ by Cloaking Intrinsic Human Serum Albumin.

Nanomaterials have become increasingly promising for biomedical applications owing to their specific biological characteristics. As drug delivery vehicles, nanomaterials have to circulate in the bloodstream to deliver the encapsulated components to the target tissues. Protein corona regulation is one of the promising approaches that gives stealth capability to avoid immune response. The aim of this study was to develop molecularly imprinted polymer nanogels (MIP-NGs) capable of protein corona regulation, using intrinsic human serum albumin (HSA) and with a functional monomer, dansylamide ethyl acrylamide (DAEAm), the dansylamide group serving as a ligand for HSA. The recognition capability of HSA for MIP-NGs was investigated by isothermal titration calorimetry (ITC). The affinity of the MIP-NGs prepared with DAEAm was then compared to that of the reference MIP-NGs prepared with pyrrolidyl acrylate developed in our previous study. Furthermore, we demonstrated that the concurrent use of these two different functional monomers for molecular imprinting was further effective to construct high-affinity recognition nanocavities for HSA and to form HSA-rich protein corona in the human plasma owing to the different interaction modes of the monomers. We believe that the molecular imprinting strategy developed through the use of ligand-based functional monomer is an effective strategy to create artificial molecular recognition materials.

Orientationally Fabricated Zwitterionic Molecularly Imprinted Nanocavities for Highly Sensitive Glycoprotein Recognition.

Glycoprotein recognition has recently gained a lot of attention, since glycoproteins play important roles in a diverse range of biological processes. Robustly synthesized glycoprotein receptors, such as molecularly imprinted polymers (MIPs), which can be easily and sustainably handled, are highly attractive as antibody substitutes because of the difficulty in obtaining high-affinity antibodies specific for carbohydrate-containing antigens. Herein, molecularly imprinted nanocavities for glycoproteins have been fabricated via a bottom-up molecular imprinting approach using surface-initiated atom transfer radical polymerization (SI-ATRP). As a model glycoprotein, ovalbumin was immobilized in a specific orientation onto a surface plasmon resonance sensor chip by forming a conventional cyclic diester between boronic acid and cis-diol. Biocompatible polymer matrices were formed around the template molecule, ovalbumin, using SI-ATRP via a hydrophilic comonomer, 2-methacryloyloxyethyl phosphorylcholine, in the presence of pyrrolidyl acrylate (PyA), a functional monomer capable of electrostatically interacting with ovalbumin. The removal of ovalbumin left MIPs with binding cavities containing boronic acid and PyA residues located at suitable positions for specifically binding ovalbumin. Careful analysis revealed that strict control over the polymer significantly improved sensitivity and selectivity for ovalbumin recognition, with a limit of detection of 6.41 ng/mL. Successful detection of ovalbumin in an egg white matrix was demonstrated to confirm the practical utility of this approach. Thus, this strategy of using a polymer-based recognition of a glycoprotein through molecularly imprinted nanocavities precisely prepared using a bottom-up approach provides a potentially powerful approach for detection of other glycoproteins.

Site-specific post-imprinting modification of molecularly imprinted polymer nanocavities with a modifiable functional monomer for prostate cancer biomarker recognition.

Recognition of glycans of glycoproteins using biotic materials such as antibodies is challenging due to lack of antigenicity. Polymeric materials suitable for the molecular recognition of glycoproteins have attracted considerable attention. In this study, we aimed to develop abiotic molecular materials for the recognition of prostate-specific antigen (PSA), a known biomarker for prostate cancer. We used a non-covalent bonding-based molecular imprinting technique to introduce post-imprinting poly(ethylene glycol)-based capping agent into a low-affinity recognition cavity. Details of the binding properties of these groups were investigated to optimize their affinity and selectivity for PSA. Molecularly imprinted polymers (MIPs) were prepared using a bottom-up approach based on surface-initiated atom transfer radical polymerization from a PSA-conjugated sensor chip with a functional monomer-bearing carboxy and secondary amine groups as interaction and post-imprinting modification (PIM) sites, respectively. PSA was orientationally conjugated on the sensor chip through diesters between the immobilized 3-fluorophenyl boronic acid and the cis-diol groups of PSA glucans. Treatment with the capping agent selectively inactivated low-affinity recognition cavities while protecting high-affinity cavities with the addition of a low concentration of PSA as a dynamic protection agent. The MIP thickness is critical in the present molecular imprinting, as a value of less than 5 nm can enable high selectivity. We believe that the proposed strategy based on a non-covalent molecular imprinting approach combined with a PIM-based capping treatment provides a novel method for the development of highly sensitive and selective glycoprotein recognition materials for use in biomarker sensing.

A Pretreatment-Free, Polymer-Based Platform Prepared by Molecular Imprinting and Post-Imprinting Modifications for Sensing Intact Exosomes.

Exosomes are small (30-100 nm) membrane vesicles that serve as regulatory agents for intercellular communication in cancers. Currently, exosomes are detected by immuno-based assays with appropriate pretreatments like ultracentrifugation and are time consuming (>12 h). We present a novel pretreatment-free fluorescence-based sensing platform for intact exosomes, wherein exchangeable antibodies and fluorescent reporter molecules were aligned inside exosome-binding cavities. Such antibody-containing fluorescent reporter-grafted nanocavities were prepared on a substrate by well-designed molecular imprinting and post-imprinting modifications to introduce antibodies and fluorescent reporter molecules only inside the binding nanocavities, enabling sufficiently high sensitivity to detect intact exosomes without pretreatment. The effectiveness of the system was demonstrated by using it to discriminate between normal exosomes and those originating from prostate cancer and analyze exosomes in tear drops.

Regulation of protein-binding activities of molecularly imprinted polymers via post-imprinting modifications to exchange functional groups within the imprinted cavity.

We prepared lysozyme-imprinted polymers bearing modifiable sites within the imprinted cavity to introduce various functional groups via post-imprinting modifications. For this purpose, ({[2-(2-methacrylamido)-ethyldithio]-ethylcarbamoyl}-methoxy)acetic acid (MDTA), which has a carboxy group to interact with the target protein, lysozyme, and a disulfide linkage for post-imprinting modifications, was used as a functional monomer. A lysozyme-imprinted polymer film was prepared by copolymerization of MDTA with a cross-linker, N,N'-methylenebisacrylamide, in the presence of lysozyme. After removing lysozyme, followed by reducing the disulfide linkage, various functional groups, such as carboxy, amino, sulfonate, and oligo-ethylene oxide, were introduced to the exposed thiol groups via a disulfide exchange reaction using the pyridyldisulfide derivatives of these functional groups. Various functional groups could be introduced reversibly via this post-imprinting disulfide exchange reaction after the construction of the lysozyme-imprinted cavities. The modification regulated the protein-binding activity. The proposed post-imprinting modification system, based on a molecular imprinting process, is expected to lead to the development of advanced materials for fine-tuning and/or introducing desired functions.

Fabrication of Redox-Responsive Degradable Capsule Particles by a Shell-Selective Photoinduced Cross-Linking Approach from Spherical Polymer Particles.

In this study, a fabrication route towards functional capsule particles was successfully developed by means of a self-templating shell-selective cross-linking strategy that enables us to prepare shell-cross-linked hollow polymer particles directly from homogeneous spherical polymer particles. To prepare redox-responsive degradable capsule particles, a newly designed monomer bearing a photoinduced post-cross-linking group (cinnamoyl group) and a redox-environment-responsive cleavable group (disulfide group), N-cinnamoyl-N'-methyacryloylcystamine (MCC), was synthesized. Redox-responsive degradable capsule particles were successfully prepared from homogeneous spherical poly(MCC)-based particles by a self-templating shell-selective photoinduced cross-linking approach. Moreover, the cargo loading capability of the shell-cross-linked hollow particles was confirmed through a solvent exchange procedure using dyes, polymer precursors and anticancer reagents. Furthermore, redox-responsive degradability of the capsule polymer particles was also confirmed by adding a reducing agent for cleavage of the disulfide linkage. We hope that the efficient fabrication route of functional capsule particles directly from spherical polymer particles opens efficient routes for the fabrication of a wide range of capsule particles; in particular, this technique is robust, productive, and facile because neither additional sacrificial template particles nor toxic solvents are required.

Regioselective Molecularly Imprinted Reaction Field for [4 + 4] Photocyclodimerization of 2-Anthracenecarboxylic Acid.

Molecularly imprinted cavities have functioned as a regioselective reaction field for the [4 + 4] photocyclodimerization of 2-anthracenecarboxylic acid (2-AC). Molecularly imprinted polymers were prepared by precipitation polymerization of N-methacryloyl-4-aminobenzamidine as a functional monomer to form a complex with template 2-AC and ethylene glycol dimethacrylate as a crosslinking monomer. The 2-AC-imprinted cavities thus constructed preferentially bound 2-AC with an affinity greater than that toward structurally related 9-anthracenecarboxylic acid, 2-aminoanthracene, and unsubstituted anthracene. Moreover, from the four possible regioisomeric cyclodimers, they mediated the [4 + 4] photocyclodimerization of 2-AC specifically to the anti-head-to-tail (anti-HT) isomer. This indicates that the imprinted cavities accommodate two 2-AC molecules in an anti-HT manner, thereby facilitating the subsequent regioselective photocyclodimerization.

Molecularly Imprinted Nanogels Acquire Stealth In†Situ by Cloaking Themselves with Native Dysopsonic Proteins.

Protein corona formation was regulated on the surface in vivo by molecular imprinting to enable polymeric nanogels to acquire stealth upon intravenous administration. Albumin, the most abundant protein in blood, was selected as a distinct protein component of protein corona for preparing molecularly imprinted nanogels (MIP-NGs) to form an albumin-rich protein corona. Intravital fluorescence resonance energy transfer imaging of rhodamine-labeled albumin and fluorescein-conjugated MIP-NGs showed that albumin was captured by MIP-NGs immediately after injection, forming an albumin-rich protein corona. MIP-NGs circulated in the blood longer than those of non-albumin-imprinted nanogels, with almost no retention in liver tissue. MIP-NGs also passively accumulated in tumor tissue. These data suggest that this strategy, based on regulation of the protein corona in vivo, may significantly influence the development of drug nanocarriers for cancer therapy.

Efficient Pathway for Preparing Hollow Particles: Site-Specific Crosslinking of Spherical Polymer Particles with Photoresponsive Groups That Play a Dual Role in Shell Crosslinking and Core Shielding.

Site-specific a posteriori photocrosslinking of homogeneous spherical polymer particles and subsequent removal of the particle core-the self-templating strategy-has been developed as an efficient pathway for hollow particle formation. In this approach, homogeneous polymer particles containing linear polymers bearing post-crosslinkable side-chain groups are first synthesized, and the photoinduced crosslinking occurred only at the shell region in the homogeneous polymer particles. Our fundamental studies clarified that the remaining non-crosslinked photoresponsive groups in the shell region played a crucial role in shielding the core region from photoirradiation. The shell-selective crosslinking was successfully applied to hollow polymer particle formation by core removal. This facile route to polymeric hollow particle formation via a self-templating strategy has great potential to be used as an alternative because the route has high mass productivity and high simplicity as a result of the non-use of additional sacrificial template particles and highly toxic solvents.

A Programmable Signaling Molecular Recognition Nanocavity Prepared by Molecular Imprinting and Post-Imprinting Modifications.

Inspired by biosystems, a process is proposed for preparing next-generation artificial polymer receptors with molecular recognition abilities capable of programmable site-directed modification following construction of nanocavities to provide multi-functionality. The proposed strategy involves strictly regulated multi-step chemical modifications: 1) fabrication of scaffolds by molecular imprinting for use as molecular recognition fields possessing reactive sites for further modifications at pre-determined positions, and 2) conjugation of appropriate functional groups with the reactive sites by post-imprinting modifications to develop programmed functionalizations designed prior to polymerization, allowing independent introduction of multiple functional groups. The proposed strategy holds promise as a reliable, affordable, and versatile approach, facilitating the emergence of polymer-based artificial antibodies bearing desirable functions that are beyond those of natural antibodies.

Molecularly Imprinted Polymer Arrays as Synthetic Protein Chips Prepared by Transcription-type Molecular Imprinting by Use of Protein-Immobilized Dots as Stamps.

Molecularly imprinted polymer (MIP) arrays were demonstrated for the recognition of proteins. They were prepared via transcription-type molecular imprinting where patterned dots composed of biotinylated nanoparticles were first immobilized on a glass substrate followed by the immobilization of versatile biotinylated proteins via avidin-biotin interactions, yielding a multiple protein-immobilized stamp as a mold that could be transcribed. MIPs were prepared between the stamp and a methacrylated glass substrate, and after the stamp was peeled off, MIP dots were able to be prepared on the methacrylated glass substrate according to the positions of the immobilized proteins on the stamp. We confirmed that the prepared MIP array showed the expected selective binding toward the corresponding template proteins by conducting competitive binding assays using the fluorescently labeled proteins as corresponding competitors. The binding behaviors were consistent with those obtained by a surface plasmon resonance sensing system. We believe that the proposed platform involving the easily handled nanoparticle-based protein stamps for the preparation of MIP arrays can provide a new type of pattern recognition-based protein chip, which can be adopted as a substitute for the use of conventional protein arrays in various research and industrial fields in the life sciences.

Amphiphilic Polymerizable Porphyrins Conjugated to a Polyglycerol Dendron Moiety as Functional Surfactants for Multifunctional Polymer Particles.

An amphiphilic polyglycerol dendron (PGD) conjugated porphyrin (PGP) bearing a polymerizable group was successfully synthesized. The PGP was used as an effective surfactant in emulsion and microsuspension polymerization systems to prepare styrene and methacrylate polymer particles, and the use of PGP provided the simple polymer particles with fluorescence derived from the metalloporphyrin and high colloidal stability due to the PGD. Furthermore, based on confocal laser scanning microscopy, we observed that the particles spontaneously formed a core-shell morphology with the PGP localized in the shell region during the polymerization and demonstrated drug loading in the shell region using rhodamine B as a model drug. The results indicate that the use of the functional surfactant PGP led to the preparation of multifunctional polymer particles from simple monomer species, and the resulting particles possessed high colloidal stability, fluorescence, and drug loading capability.

Synthesis of Monodispersed Submillimeter-Sized Molecularly Imprinted Particles Selective for Human Serum Albumin Using Inverse Suspension Polymerization in Water-in-Oil Emulsion Prepared Using Microfluidics.

We synthesized monodispersed submillimeter-sized (100 µm-1 mm) microgels by inverse suspension polymerization of water-soluble monomer species with a photoinitiator in water-in-oil (W/O) droplets formed by the microchannel. After fundamental investigations of the selection of suitable surfactants, surfactant concentration, and flow rate, we successfully prepared monodispersed submillimeter-sized W/O droplets. Because radical polymerization based on thermal initiation was not appropriated based on colloidal stability, we selected photoinitiation, which resulted in the successful synthesis of monodispersed submillimeter-sized microgels with sufficient colloidal stability. The microgel size was controlled by the flow rate of the oil phase, which maintained the monodispersity. In addition, the submillimeter-sized microgels exhibit high affinity and selective binding toward HSA utilizing molecular imprinting. We believe the monodispersed submillimeter-sized molecularly imprinted microgels can be used as affinity column packing materials without any biomolecules, such as antibodies, for sample pretreatment to remove unwanted proteins without a pump system.

Preparation of molecularly imprinted polymers for the recognition of proteins via the generation of peptide-fragment binding sites by semi-covalent imprinting and enzymatic digestion.

Molecularly imprinted polymers bearing peptide fragment-based binding sites within the protein-imprinted cavities were prepared by copolymerization of the acrylated protein with 6-monoacryloyl-trehalose and 6,6'-diacryloyl-trehalose as a hydrophilic comonomer and a crosslinker respectively, followed by enzymatic decomposition of the grafted protein into the polymer matrix with pepsin, resulting in the creation of peptide fragment-based protein-binding sites.

Synthesis of grafted phosphorylcholine polymer layers as specific recognition ligands for C-reactive protein focused on grafting density and thickness to achieve highly sensitive detection.

We studied the effects of layer thickness and grafting density of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) thin layers as specific ligands for the highly sensitive binding of C-reactive protein (CRP). PMPC layer thickness was controlled by surface-initiated activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP). PMPC grafting density was controlled by utilizing mixed self-assembled monolayers with different incorporation ratios of the bis[2-(2-bromoisobutyryloxy)undecyl] disulfide ATRP initiator, as modulated by altering the feed molar ratio with (11-mercaptoundecyl)tetra(ethylene glycol). X-ray photoelectron spectroscopy and ellipsometry measurements were used to characterize the modified surfaces. PMPC grafting densities were estimated from polymer thickness and the molecular weight obtained from sacrificial initiator during surface-initiated AGET ATRP. The effects of thickness and grafting density of the obtained PMPC layers on CRP binding performance were investigated using surface plasmon resonance employing a 10 mM Tris-HCl running buffer containing 140 mM NaCl and 2 mM CaCl2 (pH 7.4). Furthermore, the non-specific binding properties of the obtained layers were investigated using human serum albumin (HSA) as a reference protein. The PMPC layer which has 4.6 nm of thickness and 1.27 chains per nm(2) of grafting density showed highly sensitive CRP detection (limit of detection: 4.4 ng mL(-1)) with low non-specific HSA adsorption, which was improved 10 times than our previous report of 50 ng mL(-1).